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Tendinopathies account for 30% of 102 million annual musculoskeletal injuries occurring annually in the United States. Current treatments, like dry needling, induce microdamage to promote healing but produce mixed success rates. Previously, we showed focused ultrasound can noninvasively create microdamage while preserving mechanical properties in ex vivo murine tendons. This present study compared growth factor, histological, and mechanical effects after focused ultrasound or dry needling treatments in an in vivo murine tendon injury model. Partial Achilles tenotomy was performed in 26 rats. One-week postsurgery, tendons were treated with focused ultrasound (1.5 MHz, 1-ms pulses at 10 Hz for 106 s, p +  = 49 MPa, p -  = 19 MPa) or dry needling (30 G needle, 5 fenestrations over 20 s) and survived for 1 additional week. Blood was collected immediately before and after treatment and before euthanasia; plasma was assayed for growth factors. Treated tendons and contralateral controls were harvested for histology or mechanical testing. No differences were found between treatments in release of insulin growth factor 1 and transforming growth factor beta; vascular endothelial growth factor A concentrations were too low for detection. Histologically, focused ultrasound and dry needling tendons displayed localized fibroblast infiltration without collagen proliferation with no detectable differences between treatments. Mechanically, stiffness and percent relaxation of dry needling tendons were lower than controls (p = 0.0041, p = 0.0441, respectively), whereas stiffness and percent relaxation of focused ultrasound tendons were not different from controls. These results suggest focused ultrasound should be studied further to determine how this modality can be leveraged as a therapy for tendinopathies., (© 2023 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.) more...

Histotripsy fractionates most soft tissues; however, healthy tendons have shown resistance to histotripsy fractionation. Prior work has shown that pre-heating tendons increases susceptibility to histotripsy fractionation; combining multiple driving frequencies may also allow successful fractionation of tendons. Here, we evaluate single- and dual-frequency histotripsy in four healthy and eight tendinopathic ex vivo bovine tendons. First, we evaluated single-frequency (1.07, 1.5, and 3.68 MHz) and dual-frequency (1.07 and 1.5 MHz or 1.5 and 3.68 MHz) bubble dynamics with high-speed photography in a tissue-mimicking phantom. Then, tendons were treated with histotripsy. Cavitation activity was monitored with a passive cavitation detector (PCD) and targeted areas were evaluated grossly and histologically. Results in tendinopathic tendons showed 1.5 MHz or 3.68 MHz single-frequency exposure caused focal disruption, whereas 1.5 and 3.68 MHz dual-frequency exposures caused fractionated holes; all treatments caused some thermal denaturation. Exposure to 1.07 MHz alone or combined with 1.5 MHz did not show fractionation in tendinopathic tendons. In healthy tendons, only thermal necrosis was observed for all tested exposures. PCD showed some differences in cavitation activity in tendinopathic tendons but did not predict successful fractionation. These results suggest that full histotripsy fractionation is possible using dual-frequency exposures in tendinopathic tendons., (© 2023 Acoustical Society of America.) more...

Objective. High-intensity focused ultrasound (HIFU) can induce thermal and mechanical mechanisms in a well-defined focal volume of tissues. Histotripsy is a form of mechanical HIFU that can initiate and interact with bubble(s) to cause shock scattering and perhaps atomization within the bubble(s) to fractionate most soft tissues. Ultrasonic atomization, or the ejection of fine droplets from an acoustically-excited liquid exposed to air, has been shown to erode planar soft tissue surfaces, which has led to theories that atomization is a mechanism in histotripsy. However, healthy tendons show resistance to conventional histotripsy; pre-treatment of tendons with heat increases susceptibility to histotripsy fractionation. This study investigates ultrasonic atomization and erosion from planar healthy and tendinopathic tendon surfaces as we evaluate HIFU parameters for histotripsy in tendons. Approach. Forty-six ex vivo bovine tendon-air interfaces were pre-conditioned to surface wetting, heat baths of 20 °C (unaltered), 37 °C (body temperature), and 58 °C (collagen degradation), collagenase soaks for 1, 3, 5, and 24 h (mimicking tendinopathic tendons), and phosphate buffered saline soaks for 24 h. Ejected fragments, histology, and gross analysis determined erosion success. Tissue displacement from the HIFU radiation force was monitored with high-speed photography, and tissue relaxation was pixel-tracked and fit to a Kelvin-Voigt model to evaluate changes in viscoelastic properties. Main results. Results showed that atomization produced holes in 24 h collagenase tendons and surface pitting in 58 °C, 3 h, and 5 h collagenase tendons. Increased mound heights and viscoelastic constants in pre-heated (to 58 °C) and collagenase-soaking (3+ hours) tendinopathic models caused a decrease in elasticity and/or increase in viscosity, increasing susceptibility to erosion by HIFU atomization. Significance. Therefore, tendons with chronic tendinopathies may be more susceptible than healthy tendons to histotripsy fractionation., (© 2023 Institute of Physics and Engineering in Medicine.) more...

Abstract: Tendon injury is prevalent and costly in the United States, comprising 45% of the 66 million musculoskeletal injuries and costing $114 billion annually. Surgical and therapeutic methods, such as arthroscopic surgery, dry needling, and physical therapy, produce mixed success in reintroducing a healing response in tendinopathy due in part to inconsistent dosing and monitoring. Ultrasound is one therapeutic modality that has been shown to noninvasively induce bioeffects in tendon that may help promote healing. However, results from this modality have also been mixed. This review compares the current state of the field in therapeutic ultrasound and shockwave therapy, including low-intensity therapeutic ultrasound, extracorporeal shockwave therapy, and radial shockwave therapy, and evaluates the efficacy in treating tendinopathies with ultrasound. We found that the mixed successes may be attributed to the wide variety of achievable parameters within each broader treatment type and the lack of standardization in measurements and reporting. Despite mixed outcomes, all three therapies show potential as an alternative therapy with lower-risk adverse effects than more invasive methods like surgery. There is currently insufficient evidence to conclude which ultrasound modality or settings are most effective. More research is needed to understand the healing effects of these different therapeutic ultrasound and shockwave modalities., Competing Interests: Financial disclosure statements have been obtained, and no conflicts of interest have been reported by the authors or by any individuals in control of the content of this article., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.) more...

The development of tunable, ultrasound-responsive hydrogels that can deliver protein payload on-demand when exposed to focused ultrasound is described in this study. Reversible Diels-Alder linkers, which undergo a retro reaction when stimulated with ultrasound, were used to cross-link chitosan hydrogels with entrapped FITC-BSA as a model protein therapeutic payload. Two Diels-Alder linkage compositions with large differences in the reverse reaction energy barriers were compared to explore the influence of linker composition on ultrasound response. Selected physicochemical properties of the hydrogel construct, its basic degradation kinetics, and its cytocompatibility were measured with respect to Diels-Alder linkage composition. Focused ultrasound initiated the retro Diels-Alder reaction, controlling the release of the entrapped payload while also allowing for real-time visualization of the ongoing process. Additionally, increasing the focused ultrasound amplitude and time correlated with an increased rate of protein release, indicating stimuli responsive control. more...

The elastic modulus, or slope of the stress-strain curve, is an important metric for evaluating tissue functionality, particularly for load-bearing tissues such as tendon. The applied force can be tracked directly from a mechanical testing system and converted to stress using the tissue cross-sectional area; however, strain can only be calculated in post-processing by tracking tissue displacement from video collected during mechanical testing. Manual tracking of Verhoeff stain lines pre-marked on the tissue is time-consuming and highly dependent upon the user. This paper details the development and testing of an automated processing method for strain calculations using Harris corner detection. The automated and manual methods were compared in a dataset consisting of 97 rat tendons (48 Achilles tendons, 49 supraspinatus tendons), divided into ten subgroups for evaluating the effects of different therapies on tendon mechanical properties. The comparison showed that average percent differences between the approaches were 0.89% and -2.10% for Achilles and supraspinatus tendons, respectively. The automated approach reduced processing time by 83% and produced similar results to the manual method when comparing the different subgroups. This automated approach to track tissue displacements and calculate elastic modulus improves post-processing time while simultaneously minimizing user dependency., (© 2022. The Author(s) under exclusive licence to Biomedical Engineering Society.) more...

Tendon injuries are extremely common, resulting in mechanically weaker tendons that could lead to tendon rupture. Dry needling (DN) is widely used to manage pain and function after injury. However, DN is invasive and high inter-practitioner variability has led to mixed success rates. Focused ultrasound (fUS) is a non-invasive medical technology that directs ultrasound energy into a well-defined focal volume. fUS can induce thermal ablation or mechanical fractionation, with bioeffect type controlled through ultrasound parameters. Tendons must withstand high physiological loads, thus treatments maintaining tendon mechanical properties while promoting healing are needed. Our objective was to evaluate mechanical effects of DN and 3 fUS parameter sets, chosen to prioritize mechanical fractionation, on Achilles and supraspinatus tendons. Ex vivo rat Achilles and supraspinatus tendons (50 each) were divided into sham, DN, fUS-1, fUS-2, and fUS-3 (n = 10/group). Following treatment, tendons were mechanically tested. Elastic modulus of supraspinatus tendons treated with DN (126.64 ± 28.1 MPa) was lower than sham (153.02 ± 29.3 MPa; p = 0.0280). Stiffness and percent relaxation of tendons treated with DN (Achilles: 114.40 ± 31.6 N/mm; 49.10 ± 6.1%; supraspinatus: 109.53 ± 30.8 N/mm; 50.17 ± 7.6%) were lower (all p < 0.0334) than sham (Achilles: 141.34 ± 20.9 N/mm; 60.30 ± 7.7%; supraspinatus: 135.14 ± 30.2 N/mm; 60.85 ± 15.4%). Modulus of Achilles and supraspinatus tendons treated with fUS-1 (159.88 ± 25.7 MPa; 150.12 ± 22.0 MPa, respectively) were similar to sham (156.35 ± 23.0 MPa; 153.02 ± 29.3 MPa, respectively). These results suggest that fUS preserves mechanical properties better than DN, with fUS-1 performing better than fUS-2 and fUS-3. fUS should be studied further to fully understand its mechanical and healing effects to help evaluate fUS as an alternative, non-invasive treatment for tendon injuries., (Copyright © 2021 Elsevier Ltd. All rights reserved.) more...

Around 30 million tendon injuries occur annually in the U.S. costing $ 114 billion. Conservative therapies, like dry needling, promote healing in chronically injured tendons by inducing microdamage but have mixed success rates. Focused ultrasound (fUS) therapy can noninvasively fractionate tissues through the creation, oscillation, and collapse of bubbles in a process termed histotripsy; however, highly collagenous tissues, like tendon, have shown resistance to mechanical fractionation. This study histologically evaluates whether fUS mechanical disruption is achievable in tendons. Ex vivo rat tendons (45 Achilles and 44 supraspinatus) were exposed to 1.5-MHz fUS operating with 0.1-10 ms pulses repeated at 1-100 Hz for 15-60 s with peak positive pressures <89 MPa and peak negative pressures <26 MPa; other tendons were exposed to dry needling or sham. Immediately after treatment, tendons were flash-frozen and stained with hematoxylin and eosin (H&E) or alpha-nicotinamide adenine dinucleotide diaphorase ( α -NADH-d) and evaluated by two reviewers blinded to the exposure conditions. Results showed successful creation of bubbles for all fUS-treated samples; however, not all samples showed histological injury. When the injury was detected, parameter sets with shorter pulses (0.1-1 ms), lower acoustic pressures, or reduced treatment times showed mechanical disruption in the form of fiber separation and fraying with little to no thermal injury. Longer pulses or treatment times showed a combination of mechanical and thermal injury. These findings suggest that mechanical disruption is achievable in tendons within a small window of acoustic parameters, supporting the potential of fUS therapy in tendon treatment. more...

In the U.S., approximately 14 million tendon and ligament injuries are reported each year. Dry needling (DN) is a conservative treatment introduced to alleviate pain and restore function; however, it is invasive and has mixed success. Focused ultrasound (fUS) is a non-invasive technology that directs ultrasound energy into a well-defined focal volume. fUS induces thermal and/or mechanical bioeffects which can be controlled by the choice of ultrasound parameters. fUS could be an alternative to DN for treatment of tendon injuries, but the bioeffects must be established. Thus, the purpose of this pilot study was to compare the effect of DN and fUS on the mechanical properties and cell morphology of 30 ex vivo rat Achilles tendons. Tendons were randomly assigned to sham, DN, or fUS, with 10 tendons per group. Within each group, 5 tendons were evaluated mechanically, and 5 tendons were analyzed histologically. Elastic modulus in the DN (74.05 ± 15.0 MPa) group was significantly lower than sham (149.84 ± 59.1 MPa; p = 0.0094) and fUS (128.84 ± 28.3 MPa; p = 0.0453) groups. Stiffness in DN (329.05 ± 236.8 N/mm; p = 0.0034) and fUS (315.26 ± 68.9 N/mm; p = 0.0027) groups were significantly lower than sham (786.10 ± 238.7 N/mm) group. Histologically, localized necrosis was observed in 3 out of 5 tendons exposed to fUS, with surrounding tissue unharmed; no evidence of cellular injury was observed in DN or sham groups. These results suggest that fUS preserves the mechanical properties of tendon better than DN. Further studies are needed to evaluate fUS as an alternative, noninvasive treatment modality for tendon injuries., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.) more...

Tendon injuries comprise 45% of the 66 million musculoskeletal injuries each year with annual costs of $114 billion. Conservative therapies to treat injured tendon produce mixed success rates. Histotripsy is a high intensity focused ultrasound modality that creates and oscillates bubbles to fractionate soft tissues in a well-defined focal volume; however, highly collagenous tissues like tendon have proven resistant to histotripsy. The goal here is to explore acoustic parameters for mechanical fractionation of tendons. Large animal Achilles tendons were exposed to HIFU at 1.07-3.68 MHz with 0.005-20 ms pulses repeated at 1-1000 Hz for 15-60 s with acoustic pressures up to p+ = 97 MPa, p- = 27 MPa. A Verasonics research ultrasound imaging system and ATL L7-4 transducer was used to monitor bubble activity during exposure. Tendon samples were fixated and stained with Hematoxylin and Eosin (H&E) for cellular morphology analysis and alpha-nicotinamide dinucleotide diaphorase (αNADH-d) for enzymatic activity analysis. Results showed successful bubble creation in the focal region; however, only 1/90 samples showed focal coagulative necrosis histologically. Future work involves an evaluation of whether bubbles are of insufficient size or collapse strength to mechanically fractionate tendon. [Work supported by Penn State Seed Grant, NIH EB027886, and NSF DGE1255832]. more...

Information is scarce on gray whale (Eschrichtius robustus) anatomy and that of mysticetes in general. Dissection of the head of a neonatal gray whale revealed novel anatomical details of the eye, blowhole, incisive papilla with associated nasopalatine ducts, sensory hairs, and throat grooves. Compared to a similar sized right whale calf, the gray whale eyeball is nearly twice as long. The nasal cartilages of the gray whale, located between the blowholes, differ from the bowhead in having accessory cartilages. A small, fleshy incisive papilla bordered by two blind nasopalatine pits near the palate's rostral tip, previously undescribed in gray whales, may be associated with the vomeronasal organ, although histological evidence is needed for definitive identification. Less well known among mysticetes are the numerous elongated, stiff sensory hairs (vibrissae) observed on the gray whale rostrum from the ventral tip to the blowhole and on the mandible. These hairs are concentrated on the chin, and those on the lower jaw are arranged in a V-shaped pattern. We confirm the presence of two primary, anteriorly converging throat grooves, confined to the throat region similar to those of ziphiid and physeteroid odontocetes. A third, shorter groove occurs lateral to the left primary groove. The throat grooves in the gray whale have been implicated in gular expansion during suction feeding., (© 2015 Wiley Periodicals, Inc.) more...